The light from the sample may result from diffusion or fluorescence. Fluorescence microscopy is a technique that takes advantage of the phenomenon of fluorescence in order to observe various compounds. Fluorescence is the property possessed by certain bodies to emit fluorescent light by themselves.
The fluorescence of an observed compound can be primary, if the compound is fluorescent itself (e.g., chlorophyll, oil) or secondary, if the observed compound is marked with a fluorescent substance known as a fluorochrome or fluorescent marker.
In particular in cell biology, a large number of molecular events occurring at the cell surface are studied by fluorescence microscopy, such as cell adhesion, the binding of hormones to receptors in the plasma membrane, the secretion of neurotransmitters as well as membrane dynamics (endocytosis, exocytosis).
A fluorescence microscopy device usually comprises a light source for excitation, means for separating the excitation photons from the emission photons, a lens system for capturing the photons and, in general, imaging means.
Fluorescence techniques can be used with different types of microscopes, notably:                A conventional optical microscope where the excitation light may pass through the sample or the lens. In the latter case, this is epifluorescence microscopy;        A confocal microscope, for example by laser scanning that in particular enables three-dimensional images of the sample;        A total internal reflection fluorescence microscope (usually called TIRF), which uses an evanescent wave to excite the fluorescence that is of only a very shallow depth, immediately adjacent to the interface of the sample substrate (usually glass) and of the liquid medium (usually water), in which the sample is disposed. Lighting is performed by a laser beam incident at a supercritical angle to create an evanescent wave (exponentially decreasing orthogonally to the interface).        
TIRF microscopy, although currently booming and allowing precise observations, has some disadvantages. Indeed, the use of an adapted laser source is costly and the excitation field thus generated may not be homogeneous (due to interference from the coherence of the beam). In addition, lighting by the lens does not allow a homogeneous excitation and the resulting depth of penetration is not constant across the field to be observed. Moreover, there are containment losses of the excitation field related to the intrinsic light scattering by the cells.
FR-A-2943428 discloses a method of observation by fluorescence microscopy of a sample comprising fluorescent components in a liquid medium of refractive index nL arranged on a transparent support of refractive index ns, which is greater than nL and less than or equal to 1.55, and an observation device comprising a full-field immersion lens, whose numerical angular aperture, ON, is greater than or equal to 1.33 and less than or equal to ns, and a set of lenses for forming an image in at least one image plane, and which further comprises a mask arranged in the rear focal plane of the immersion lens or a conjugate plane of said rear focal plane, so as to obscure the fluorescence emission components of the sample in the angular directions in which the angle θ is less than or equal to a critical angle θc, with θc=arcsin (nL/ns), and the angle θ defined as the angle of an angular direction of the fluorescence emission relative to the perpendicular direction of the support surface on which the sample to be observed is arranged.
This observation method permits obtaining high-quality fluorescence images with a low-cost device or improving the quality of images obtained by TIRF microscopy.
The device and the method described above lead to very satisfactory images with good resolution. Nevertheless, in certain circumstances, one may wish to improve the resolution.